TGF-xcex2 superfamily members signal through activation of transmembrane serine-threonine kinase receptors. These receptors phosphorylate and activate Smads, a novel class of signal transducers. Signals initiated by TGF-xcex2 superfamily members are important for regulating cellular processes, including cell division, survival, differentiation, and specification of developmental fate throughout the growth and development of diverse organisms.
During early embryogenesis of the frog Xenopus laevis, the TGF-xcex2 growth factor family plays a central role in the specification and patterning of various tissues: TGF-xcex2 superfamily members activin, Vg-1, and TGF-xcex2 all induce a full range of dorsal and ventral mesodermal markers in early embryonic tissue, whereas other TGF-xcex2 superfamily members specify axial pattern or epidermal, as opposed to neural, tissue. Almost all the critical patterning events in early Xenopus embryogenesis appear to involve members of the TGF-xcex2 superfamily.
The transforming growth factor xcex2 (TGF-xcex2) superfamily of cytokines, which includes bone morphogenic proteins (BMPs), activin, TGF-xcex2, and Vg-1, regulate a wide range of normal and pathological biological processes. These processes include cell specification during development, terminal differentiation of many cell types, fibrosis during wound healing or organ damage (e.g., cirrhosis), proliferation and invasiveness of normal and transformed cells, and angiogenesis and immune suppression induced by tumors (Roberts and Sporn, Peptide growth factors and their receptors I, eds. Sporn and Roberts, Berlin, Springer-Verlage, 419-473, 1990; Sporn et al., Science 33: 532-534, 1986). For example, one member of the family, TGF-xcex2, is secreted by a wide variety of tumors and has a wide variety of immunosuppressive effects, including the ability to induce apoptosis in B and T lymphocytes (Brabletz et al., Mol. Cell Biol. 13: 1155-1162, 1993; Cahouchi et al., Oncogene 11: 1615-1622, 1995; Weller et al., Exp. Cell Res. 221: 395-403, 1995). The ability to manipulate specific aspects of TGF-xcex2 superfamily signalling in vivo would be a powerful tool both for understanding the role of these factors in normal embryonic patterning and for controlling a broad range of pathological processes.
We have discovered methods and reagents for identifying compounds that modulate TGF-xcex2 superfamily signalling. These methods and compounds are useful for the detection and treatment of conditions involving abnormal TGF-xcex2 superfamily signalling.
In the first four aspects, the invention provides methods for detecting compounds capable of modulating TGF-xcex2 superfamily signalling. The methods include the steps of providing a cell having a reporter gene operably linked to a DNA-binding-protein recognition site, in addition to having either:
a) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety,
b) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a gene activating moiety,
c) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety, or
d) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a gene activating moiety; exposing the cell to the compound; and measuring reporter gene expression in the cell, where a change in the reporter gene expression indicates that the compound is capable of modulating TGF-xcex2 superfamily signalling.
In the fifth, sixth, seventh, and eighth aspects, the invention features a cell useful for detecting a compound capable of modulating TGF-xcex2 superfamily signalling, the cell having a reporter gene operably linked to a DNA-binding-protein recognition site in addition to having either:
a) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety,
b) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a gene activating moiety,
c) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety, or
d) a first fusion gene capable of expressing a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion gene capable of expressing a second fusion protein, the second fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a gene activating moiety.
In preferred embodiments of the first eight aspects of the invention, a decrease in reporter gene expression indicates a compound that is capable of inhibiting TGF-xcex2 superfamily signalling, and an increase in reporter gene expression indicates a compound that is capable of enhancing TGF-xcex2 superfamily signalling. In other embodiments of these aspects of the invention, reporter gene expression may be assayed by a color reaction or assayed by cell viability. In still another embodiment of the first eight aspects of the invention, the cell may be a yeast cell.
In the ninth, tenth, eleventh, and twelfth aspects, the invention provides a method for detecting a compound capable of modulating TGF-xcex2 superfamily signalling. The method comprises the steps of providing a first polypeptide comprising a polypeptide fragment of FAST-1, providing a second polypeptide, the second polypeptide comprising a polypeptide fragment of either Smad2 or Smad3 (or alternatively, providing a first polypeptide comprising a polypeptide fragment of Smad2 or Smad3, and providing a second polypeptide comprising a polypeptide fragment of FAST-1), exposing the first polypeptide to the second polypeptide and to the compound, and measuring the level of interaction between the first polypeptide and the second polypeptide, wherein an alteration in the level of interaction indicates that the compound is capable of modulating TGF-xcex2 superfamily signalling.
In one preferred embodiment of the ninth, tenth, eleventh, and twelfth aspects of the invention, at least one of the first polypeptide or the second polypeptide is immobilized on a solid-phase substance. In another preferred embodiment, a decrease in the level of interaction indicates that the compound is capable of inhibiting TGF-xcex2 superfamily signalling, and an increase in the level of interaction indicates that the compound is capable of enhancing TGF-xcex2 superfamily signalling. In other embodiments of the ninth, tenth, eleventh, and twelfth aspects, the first polypeptide is produced by a cell that contains a first fusion gene capable of expressing the first polypeptide, and the second polypeptide is produced by a cell that contains a second gene capable of expressing the second polypeptide.
In various preferred embodiments of all of the above aspects of the invention, the polypeptide fragment of FAST-1 consists of, at maximum, Xenopus FAST-1 amino acids 380 to 506, human FAST-1 amino acids 234 to 365, and mouse FAST-1 amino acids 309 to 398. In other preferred embodiments of all of the aspects of the invention, the polypeptide fragment of Smad2 consists of, at maximum, Smad2 amino acids 248 to 467 or 274 to 467, and the polypeptide fragment of Smad3 consists of, at maximum, Smad3 amino acids 230 to 446, amino acids 253 to 446, amino acids 230 to 424, or amino acids 253 to 424.
In the thirteenth aspect, the invention features a polypeptide comprising a polypeptide fragment of FAST-1. In a preferred embodiment of this aspect of the invention, the polypeptide fragment of FAST-1 includes, at maximum, Xenopus FAST-1 amino acids 380 to 506, or fragments thereof, human FAST-1 amino acids 234 to 365, or fragments thereof, or mouse FAST-1 amino acids 309 to 398, or fragments thereof.
In the fourteenth, fifteenth, sixteenth, and seventeenth aspects, the invention features a method for detecting a compound capable of modulating TGF-xcex2 superfamily signalling, comprising providing a reporter gene operably linked to a DNA-binding-protein recognition site and additionally providing either:
a) a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a gene activating moiety,
b) a first fusion protein comprising a polypeptide fragment of Smad2 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety,
c) a first fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a gene activating moiety, or
d) a first fusion protein comprising a polypeptide fragment of Smad3 covalently bonded to a binding moiety capable of specifically binding to the DNA-binding-protein recognition site and a second fusion protein comprising a polypeptide fragment of FAST-1 covalently bonded to a gene activating moiety;
exposing the first fusion protein to the second fusion protein, to the reporter gene, and to the compound; and measuring the reporter gene expression, a change in the reporter gene expression indicating a compound that is capable of modulating TGF-xcex2 superfamily signalling.
In various preferred embodiments of the fifteenth, sixteenth, seventeenth, and eighteenth aspects, a change in reporter gene expression that is a decrease indicates a compound that is capable of inhibiting TGF-xcex2 superfamily signalling, and a change in the reporter gene expression that is an increase in the reporter gene expression indicates a compound that is capable of enhancing TGF-xcex2 superfamily signalling. In other embodiments, the polypeptide of FAST-1 includes Xenopus FAST-1 amino acids 380 to 506, or fragments thereof, human FAST-1 amino acids 234 to 365, or fragments thereof, or mouse FAST-1 amino acids 309 to 398, or fragments thereof; the polypeptide fragment of Smad2 includes Smad2 amino acids 248 to 467, or fragments thereof; and the polypeptide fragment of Smad3 includes Smad3 amino acids 230 to 424, or fragments thereof. In yet another embodiment, providing the first fusion protein comprises providing a first fusion gene capable of expressing the first fusion protein and providing the second fusion protein comprises providing a second fusion gene capable of expressing the second fusion protein.
In the nineteenth aspect, the invention provides a method for diagnosing a mammal having or likely to develop a disorder involving abnormal TGF-xcex2 superfamily signalling. The method includes determining whether the mammal has a mutation in a gene encoding FAST-1. In a preferred embodiment of this aspect, the mutation is in the Smad Interaction Domain (SID).
In the twentieth aspect, the invention provides methods for diagnosing a mammal having or likely to develop a disorder involving abnormal TGF-xcex2 superfamily signalling comprising determining whether the mammal has an altered level of expression of FAST-1.
In preferred embodiments of the nineteenth and twentieth aspects of the invention, the disorder is a developmental disorder, and the mammal is a human, and may be a fetus.
In the twentieth aspect, the invention features a substantially pure mammalian FAST-1 protein or polypeptide fragment thereof for use in modulating TGF-xcex2 superfamily signalling.
In preferred embodiments of the twentieth aspect, the protein or polypeptide fragment may be from a human or a rodent. In other preferred embodiments, the polypeptide fragment comprises the Smad Interaction Domain (SID). In still another preferred embodiment, the polypeptide fragment binds to Smad2 or Smad3.
In a twenty-first aspect, the invention features a substantially pure polypeptide fragment comprising the Smad Interaction Domain (SID) of FAST-1 from Xenopus, for use in modulating TGF-xcex2 superfamily signalling.
In related, twenty-second, twenty-third, and twenty-fourth aspects, the invention features substantially pure polypeptides or fragments thereof having about 50% or greater amino acid sequence identity, about 75% or greater amino acid sequence identity, and about 90% or greater amino acid sequence identity to the comparable amino acid sequence of the mammalian FAST-1 protein or polypeptide fragment thereof. Preferably, the identity is determined by comparison with the FAST-1 SID (i.e., FAST-1 amino acids 380 to 509 of Xenopus FAST-1, amino acids 234 to 365 of human FAST-1, or amino acids 309 to 398 of mouse FAST-1). In another preferred embodiment, the polypeptide fragment binds to Smad2 or Smad3.
In a twenty-fifth aspect, the invention features a substantially pure nucleic acid encoding a mammalian FAST-1 protein or polypeptide fragment thereof.
In a twenty-sixth aspect, the invention features a vector containing a nucleic acid of the twenty-fifth aspect, capable of directing expression of the protein or polypeptide fragment thereof.
In a twenty-seventh aspect, the invention features a substantially pure nucleic acid encoding a FAST-1 Smad Interaction Domain (SID).
In a twenty-eighth aspect, the invention features a cell containing the vector of the twenty-sixth and twenty-seventh aspects above.
In a twenty-ninth aspect, the invention features a method of modulating TGF-xcex2 superfamily signalling in a cell, comprising providing a cell intracellularly with a substantially pure FAST-1 protein, or polypeptide fragment thereof, wherein the FAST-1 protein or polypeptide fragment is sufficient to modulate TGF-xcex2 superfamily signalling in a cell.
In a thirtieth aspect, the invention features a method of modulating TGF-xcex2 superfamily signalling in a cell, comprising introducing, into a cell, a vector comprising a nucleic acid encoding FAST-1 protein, or polypeptide fragment thereof, wherein the vector is capable of directing expression of the protein or polypeptide fragment in a cell containing the vector, and wherein expression of the FAST-1 protein or polypeptide fragment is sufficient to modulate TGF-xcex2 superfamily signalling in a cell.
In preferred embodiments of the twenty-ninth and thirtieth aspects, the signalling may be decreased or increased.
xe2x80x9cReporter genexe2x80x9d means any gene that encodes a product whose expression is detectable. Such genes include, without limitation, lacZ, amino acid biosynthetic genes, for example, the yeast LEU2, HIS3, LYS2, TRP1, or URA3 genes, nucleic acid biosynthetic genes, the mammalian chloramphenicol transacetylase (CAT) gene or GUS gene, or any surface antigen for which specific antibodies are available. Reporter genes may encode any enzyme that provides a phenotypic marker, for example, a protein that is necessary for cell growth or a toxic protein leading to cell death, or gene encoding a protein detectable by color assay or whose expression leads to an absence of color. Other preferred reporter genes are those encoding fluorescent markers, such as the green fluorescent protein (GFP)-encoding gene, or reporter genes encoding enzymes whose activity may be detected by chemiluminescence, such as luciferase. Reporter genes may facilitate either a selection or a screen for reporter gene expression, and quantitative differences in reporter gene expression may be measured as an indication of interaction affinities.
xe2x80x9cCovalently bondedxe2x80x9d means that two domains are joined by covalent bonds, directly or indirectly. That is, the xe2x80x9ccovalently bondedxe2x80x9d proteins or protein moieties may be immediately contiguous or may be separated by stretches of one or more amino acids within the same fusion protein.
xe2x80x9cProteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d means any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
xe2x80x9cSmad2 protein or polypeptide fragment thereofxe2x80x9d means a Smad2 protein (or polypeptide fragment or domain thereof) found in Xenopus or mammalian (e.g. mouse or human) cells. A preferred domain of Smad2 is the Mad Homology 2 (MH2) domain (i.e., amino acids 274 to 467 of human or Xenopus Smad2). Also preferred are polypeptide fragments comprising the MH2 domain, that consist of, at maximum, amino acids 274 to 467 or amino acids 248 to 467 of human or Xenopus Smad2, or the corresponding amino acids that comprise Smad2 MH2 domains from other species. These polypeptide fragments are capable of interacting with the FAST-1 Smad Interaction Domain (SID).
xe2x80x9cSmad3 protein or polypeptide fragment thereofxe2x80x9d means a Smad3 protein (or polypeptide fragment or domain thereof) found in Xenopus or mammalian (e.g. mouse or human) cells. A preferred domain of Smad3 is the Mad Homology 2 (MH2) domain (i.e., amino acids 253 to 446 of human Smad3). Also preferred are polypeptide fragments comprising the MH2 domain, that consist of, at maximum, human Smad3 amino acids 230 to 446, and subfragments thereof, consisting of, at maximum, amino acids 253 to 446, amino acids 253 to 424, or amino acids 230 to 424, or the corresponding amino acids that comprise Smad3 MH2 domains from other species. These polypeptide fragments are capable of interacting with the FAST-1 SID domain.
xe2x80x9cMammalian FAST-1 protein or polypeptide fragment thereofxe2x80x9d means an amino acid sequence derived from a mammalian cell which displays at least 30%, preferably, 40%, more preferably 50%, still more preferably 60%, 70%, or even 80% means amino acid sequence identity to a FAST-1 Smad Interaction Domain (SID), i.e., amino acids 380 to 506 of the Xenopus FAST-1 protein, amino acids 234 to 365 of the human FAST-1 protein, or amino acids 309 to 398 of the mouse FAST-1 protein. The length of comparison, generally will be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 30 amino acids. Preferably, a mammalian FAST-1 protein, or polypeptide fragment thereof, is able to bind Smad2. The FAST-1 SID is a preferred polypeptide fragment of FAST-1.
xe2x80x9cOperably linkedxe2x80x9d means that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
xe2x80x9cBinding moietyxe2x80x9d means a stretch of amino acids which is capable of directing specific polypeptide binding to a particular DNA sequence (i.e., a xe2x80x9cprotein binding sitexe2x80x9d).
xe2x80x9cModulatory compoundxe2x80x9d or xe2x80x9cmodulating compoundxe2x80x9d, as used herein, means any compound capable of either increasing or decreasing the amount of signalling initiated by a TGF-xcex2 superfamily member.
xe2x80x9cSubstantially pure proteinxe2x80x9d or substantially pure polypeptidexe2x80x9d means a protein or polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a xenopus or mammalian, e.g. human or mouse, FAST-1 polypeptide, or polypeptide fragment thereof, that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure mammalian, e.g. human or mouse, FAST-1 polypeptide, or polypeptide fragment may be obtained, for example, by extraction from a natural source (e.g. a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding a FAST-1 polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.
xe2x80x9cSubstantially pure nucleic acidxe2x80x9d means nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant nucleic acid that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic nucleic acid of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cSubstantially identicalxe2x80x9d means a polypeptide or nucleic acid exhibiting at least 75%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Default settings of sequence analysis software programs employ parameters that are considered, by those of skill in the art, to yield biologically significant results; i.e., an alignment of two polypeptides that shows one or more amino acid stretches having a high percentage of sequence identity represents two polypeptides that share a functional relationship. For example, FAST-1 polypeptides are identified by virtue of their possessing an amino acid sequence that displays at least 30% identity to a FAST-1 SID.